Production of antibodies



Patented Nov. 23, 1948 2,454,752 PRODUCTION OF ANTIBODIES? Arthur F.Coca, ;radell. N. J assignor. by mesne assignments. to American CyanamidCompany, New York, N, Y.,, a corporation of Maine No Drawing.

This invention relatesto. the production of a t odi s, more particularlyto -the pr p t of blood serum containing agglutinins. of value n thidentification and. classification of blood groups and blood types.

It has now been firmly established that the blood of higher animals;contains certain antigenic substances and: antibodies, such as,specifically, agg-lutinogens and agglutini-ns; Early w-ork demonstratedthe fact: that human blood contained two distinct hema-agglutinogenswhich were called A and B: and two hema-agglutinins which were. calledanti-A and anti-B. Corresponding agglutinogens and agglutinins do notcot-exist in the blood. of. one. individual. It has been possible,therefore, to.- group: the blood of human beings into; one of fourclasses, namely, group- A, which blood: contains the, agglutinogen A andthe agglutinin anti-B; group 13, containing agglutirrogen B andagglutinin. anti-A; group AB, which blood contains agglutinogens A and Bbut no, agglutinins; and group 0., whi-ch contains no agglutinogens butagglutim'ns anti-A and anti- B. The agglutinogens are found in; theerythros cytes; andtissue cells: whereas the agglutinins are found intheserum...

When the blood of. a donor of group Ais-introduced. into the bloodstream of an individual of group B" or 0, during a, blood transfusionthe agglutinin anti-A of the groupB, or 0, blood will agglutinate or.hemolyze the erythrocytes of. the donor blood: and a serious accidentmay' occur. Similarly, hemolysis may occur: in the-event that the donorhasblood or group B and the recipient has bloodof group A 017-0, Sincethe blood serum of; blood of group Q contains bQth-s agglutinins anti-Aand anti B itwould: appear that blood of group. 0- could not beused; asdonor blood in transfusions to; patients of" groups A-, B, or AB.However; as. thevolumeot blood; donated. is relatively scall and as? therecipientuhasa substantial proportion of. the: substance; A on; B, inhis; body tissueszin. addition, to-that. in. his;b1ood';,neutraliization of the anti-A or anti-B takes place in mostcases without seriousd-ifliculty.

Although the relative distribution. of the agglutinins anti-A, andanti-B.- varies somewhat. with racial. groups. it. hasv been. foundthat. the blood of. approximately 42% of human beings in the UnitedStates contains; the: agglutinin anti-B, 10.9% of; human beings have'blood containingthe ag lutinin anti-A, and; approximately 45%. haveblood with both anti -Al and, anti-13.. It isapparent, therefore, that,it. is ot considerable-practical importance to be: able toldetmmine raidly and Application December 20, 1944, Serial No. 569,112.

11. Claims. (Cl..167'78) accurately the blood groups of individuals sothat blood transfusions may be given when needed without unnecessarydelay and without danger of untoward reactions.

Other antigenic factors, such as M and N, have been reported but. asthese antigens do not have the ability to cause isoimmunization they arenot considered of significance in blood transfusions. They areimportant, however, in blood typing in determining parentage inmedico-legal cases. A group of miscellaneous, unidentified antigens hasbeen: designated as group X, but this group, too, is usuallydisregarded. in grouping blood for transfusions.

Withirr thevpast few years another important antigenic substance hasbeen discovered in human blood This factor was originally discovered in:the bloodof the Rhesus monkey and has been designated: at the Rh factor.This antigenic substance has the abiilty to cause isoimmunization andis, therefore, an important factor to be taken intoconsideration whenblood transfusions are to. be made. Approximately 87% of the individualsin this country have been found to have the Rh" factor in their bloodwhereas the remaining 13% are Rh negative. Since the Rh antigen is anagglutinogen, it will. give rise to the production of agglutinins andthere is the grave possibility that those individuals who have Rhnegative blood may become isoimmunized against the 'Rhfactor and may,during the course of a subsequent. blood transfusion from a Rh positivedonor, suffer a reaction which may be fatal.

It has been suspected for sometime and now seems to be fairly wellestablished that most cases oil erythroblastosis fetalis are caused byisoimmunization. For example, the mother may boot the lit-h negativetype whereas the fetus is, through heredity, Rh positive. Because ofsome defect in the placenta, someof the fetal blood may enter the bloodstream, of the mother, giving rise to anti-Rh;agglutininswhich then passback into the fetus to cause hemolysis of its red; blood cells.

The mothers blood in such circumstances may contain antirRh agglutinins,and in the event that she requires ablood transfusion and if the blooddonor is Rh positive, a severe accident may It, is obviously necessary,therefore, to provide-a rapid and effective method of determiningthe'bloodtypes Rh and anti-Rh.

Methods ofgrouping blood of the A, B, AB and 0 types havebeen developedand used for some time. Unfortunately, the production of typing serumisnot arr-easy matter and the product has m t been of? the potencydesired. The typing anti-Rhserum and blood be placed in a warm waterbath at 37 C. for about an hour, then centrifuged, resuspended ifnecessary and the results determined with the aid of a hand lens.

It isquite apparent that this delicate test is not I suitable forgeneral use. 7

In the production of the testing sera it is necessary to obtain a supplyof serum containing agglutinins of the proper type, anti-A, anti-B,anti- Rh, or the like, but free from undesirable agglu.

tinins which might interfere with the test. For example, grouping serumfor testing for blood of Group A should contain the agglutinin anti A,but none of anti-B, anti-Rh or anti-X. To obtain such serum it is firstnecessary to obtain a supply of blood with the proper agglutinin in it.This may be accomplished by injecting the appropriate antigen into theblood stream of a warm-blooded animal and, after agglutinins have beenformed, bleeding the animal.

To obtain blood serum containing anti-A it has been the practice toinject human A cells into one of the higher animals, preferably arabbit. During the course of a week or two the rabbit develops theagglutinin anti-A and its blood may be withdrawn and the serum used inthe production of blood typing substance. Unfortunately, humanerythrocytes also contain other antigenic substances which give rise tothe production of corresponding agglutinins in the rabbits blood stream.These antigens, or agglutinogens, are known collectively as factor X andit happens that the X antigens are more potent than the A antigens instimulating the production of antibodies. As a result, the relativelystrong antigen X competes unfavorably with the antigen A in theimmunization and the rabbit blood contains a relatively high proportionof the agglutinin anti-X and less than could be desired of anti-A. Inmost animals, and in fact in most of the rabbits used in this method ofproducing agglutinins, the yield of anti-A, and also anti-B and anti-Rh,is too small to be of any use. When the rabbit blood contains a suitablyhigh potency of anti-A, it may be used only after removing the anti-Xagglutinin. This is usually accomplished by a process called absorption,or neutralization, in which the anti-X factor in the rabbit serum isabsorbed by the X factor in blood cells and are thus removed from thefluid. It has usually been considered necessary to first dilute theserum with ten to twenty volumes of saline and then mix ten to twentyvolumes of blood of group O with each volume of the immunized rabbitserum. As will be seen, the production of blood grouping sera requiresthe use of excessive amounts of blood and usually fails to give aproduct of the potency desired,

I have discovered that blood grouping sera containing agglutinins inmuch higher potency than those previously thought possible can beobtained by the process to be'described and claimed herein which dependsupon the principle of blocking out undesirable antigens from the bloodmaterial used in creating agglutinins of the desired type. For example,to produce blood grouping serum containin a relatively highagglutination power for blood of any desired group I employ in theimmunization blood cells in which the undesirable, antigenic substanceswhich might interfere are rendered impotent by neutralization orotherwise to produce antibodies.

As pointed out before, it is the usual practice to inject rabbits withblood containing the agglutinogen A, or B, with the expectation that theagglutinin anti-A, or anti-B, will be produced.

This method does not take into account the disturbing influence of themore potent agglutinogen X which simultaneously causes the production ofanti-X in the rabbit blood to the detriment of the production of anti-A,or anti-B. To overcome this detrimental action, I first block out orinactivate the antigenic substance or substances collectively known asX. This is done by treatment of the A blood with a suitable blockingserum containing suflicient anti-X to neutralize the factor X. Theresulting blood cells with factor X relatively inactive but containingfactor A, or B as the case may be, may then be injected into rabbits orother suitable warm-blooded anifuels with the result that a much higherproduction of anti-A, or B, is obtained in the blood stream of theimmunized animal. Blood from the so immunized animal may then berecovered and processed to obtain serum having a much greater potency ofanti-A, or anti-B, but containing greatly reduced amounts of otherinterfering agglutinins.

To obtain suitable blockin sera for use in the process of the presentinvention, I inject rabbits or other warm-blooded animals with washederythrocytes of human blood of group 0. After the lapse of a suitableperiod of time the agglutinin anti-X is developed in the blood stream ofthe animal and may be withdrawn and processed to obtain serum with arelatively high concentration of anti-X. When this serum is mixed withnormal blood containing erythrocytes having the agglutinogens A or B orX, the agglutinogen X is neutralized, leaving the agglutinogens A or Bin practically undiminished potency.

When preparing blood sera for testing for the Rh factor, in accordancewith the present invention, the same general principle described aboveis applicable. It is necessary, however, to make provision also forblocking out the agglutinogens A and B. Accordingly, the blocking serumthat is produced will contain the agglutinins anti-A, anti-B; and anti-Xso that the blood substance used in injecting animals to bring about theformation of anti-Rh contains no other important agglutinogen other thanthe factor Rh.

In order that my invention may be better understood, the followingexamples are given which illustrate in detail a preferred process ofobtaining blood grouping sera for the groups A, B and Rh. It will beunderstood, of course, that these examples are given primarily by way ofillustration and are not intended to limit'my invention to the exactprocedure and details described.

Example 1 The following example is directed to the production of serumcontaining a high potency of the agglutinin anti-A. As a preliminarystep, suitable blood blocking serum is first prepared. This is ,done byobtaining a supply of human blood cells of group O which cells containthe agglutinogen X but do not contain the agglutinogens Aand B. Theerythrocytes are washed twice with an equal volume of sterile,physiological saline. Healthy rabbits are then givenlan ensureintravenous injection of cc. of the washed cells in 1 cc. of salinesolution. After intervals of three or four days the rabbits are againinjected with the washed cells as before. These injections are repeatedfor a total of three to five times. Six or seven days after the lastinjection the animal is bled and the serum recovered. The resultingblood serum will be found to contain a relatively high concentration ofthe agglutinin anti-X but with none of the agglutinins anti-A or anti-B.The filtered serum may be frozen and stored for some time withoutpreservatives until ready ior use.

Antigenic substance for the production of anti-A is pre ared byobtaining a supply of erythrocytes of group A. For ordinary bloodgrouping Work pooled group A blood is preierred as it contains both ofthe sub groups A1 and A2. When preparing blood grouping reagentsspecific to groups A1, A2, etc. blood from these groups is used. Thesterile erythrocytes are washed twice with an equal volume of sterilesaline in a centrifuge. To the Washed cells is added an equal volume ofthe blocking serum prepared as described above and two volumes ofsaline. Ordinarily it will be found that this amount of blocking serumsaturates the X antigens which may be present in the group A cells.()bviously, more or less of the blocking serum may be used, dependingupon the relative amounts of factor X in the group A cells and theamount of anti-X in the blocking serum. As the A cells do not absorb allof the anti-X, it is usually necessary to add more than the theoreticalamount of the anti-X than the antigen X present would indicate.

Healthy rabbits are then given an intravenous injection with 2 cc. ofthe antigenic A blood substance just described as a first sensitizingdose. After about five days the rabbits are again given a subcutaneousinjection of 2 cc. of the mixture. Two hours later they are given a 2cc. intravenous injection of the mixture. The subcutaneous andintravenous injections are repeated three times at three to fivedayintervals.

The animal is then bled 50 cc. one week after the last injection.

Occasionally some rabbits will not produce antibodies in the desiredmanner and such animals are eliminated from further use. The remainingrabbits may be given subsequent injections with subsequent bleedingsuntil they have lost their value for such production.

The resulting blood will be found to contain a relatively high potencyof the agglutinin anti-A and relatively low potencies of anti-X oranti-B. If these latter aggiutinins are found to be presout, they may beremoved from the undiluted serum by absorption with three or fourvolumes of a mixture of B and 0 cells by treatment at room temperaturewith constant but slight agitation for about fifteen minutes. The bloodserum containing anti-A may then be recovered and assayed for potency bymethods known to those skilled in the art.

After determination of the potencyof theserum it is ready for. use. Itis usually. advisable to adjust the titer of the serum to a fixed valueso, that comparable results may be. obtained when using the product fortesting for group A. This may be accomplished by diluting the serum withsaline to a given potency. When it is desired to prepare the product inthe form of a powder for storage and use, sucrose, sodium chloride, orsome other inert diluent which has no deleterious died on. th a glut naton o blee m be d 6 eltherj before or after the product is dried.Dyestuffs may also be incorporated in the mixture to serve as a means ofidentification apart from the label on the product. The usual method ofdrying is to first freeze the product and then evaporate the Watertherefrom while maintaining the product in a frozen state in the mannerlgnown to those in the art. The dried product .may then be pulverizedand is ready for use.

same 2 Ezcample 3 This example describes the production of anti- Rhserum. Goats are used as the immunizing animal since they have beenfound to be particularly suitable for the production of anti-Rh serum ofhigh otency.

As in the preceding examples, preliminary steps involve the productionof an immunizing substance in which the antigenic material of thedesired type predominates-in this case the agglutinogenic substance Rh.This anti-genie substance is obtained by treating the blood cells of theRhesus monkey with agglutinins which neutralize the antigenic substancesother than the factor Rh which are found in the erythrocytes of theRhesus monkey. As this kind of blood contains the agglutinins A, B andX, the blocking serum will have the agglutinins anti-A, anti- B, andanti-X.

Suitable blocking serum for the production of anti-Rh is prepared byobtaining a quantity of human erythrocytes which are positive forfactors A and B but preferably negative for factor Rh. These blood cellsare Washed three times with sterile, normal saline and are thenresuspended in saline, each 1 cc. of cell sediment being suspended in 4cc. of the saline solution. The suspension is then placed in a waterbath for forty minutes at 5660 C., then removed, and

5. cc. of distilled water'for each cc. of the cell suspension is added.After holding at room temperature for hour, 2.06 cc. of 10% sodiumchloride is added for each cc. of the cell suspension to give a productcontaining 1% of sodium chloride. The suspension is then centrifuged,the. supernatant liquid removed and the sediment, which is stroma, isrecovered.

Healthy goats are then given a subcutaneous injection of 10, co. in eachhind leg of an immunizing substance containing 40 cc. of the stromadescribed above containing approximately, equal parts of B-Rh negativecells and A-Rh negative cells, 160 cc. of mineral oil, 40 cc. of Falbabase (lanolin derivative) and a very small amount of a phenolmercuroborate preservative. The injection is repeated after ten days.After another ten days the goat is given a subcutaneous injection of 3cc. of the stroma in 3 cc. of saline solution. Four. hours later theanimal is given an intravenous injection of 1.5 cc. of the stroma in 3-cc. of saline. After three days the sub cutaneous and intravenousinjections arev repcated. Seven days after the last injection thevanimal may be bled to obtain a suitable blocking serum.

Rhesus monkey cells are washed three times with sterile, physiologicalsaline solution and then saturated with the blocking serum justdescribed.

Enough of the blocking serum is added so that the supernatant liquidcontains a small excess of anti-A, anti-B, and anti-X. The mixture ofblocking serum and monkey cells is then allowed to stand in an ice boxovernight after which the supernatant liquid is removed and the cellsare frozen. On thawing, the mass becomes fluid and easily injectable.Each goat is then injected five times at ten day intervalsintramuscularly with about 63 cc. of a mixture of cc. of the saturatedmonkey cells, 25 cc.-of aluminum cream, and 13 cc. of saline.

Following the immunization, the animal is bled and the serum recovered.Serums having a high potency of agglutinins effective against the Rhfactor may be used directly in blood typing.

moving traces of anti-A, anti-B and anti-X by treatment with normalanimal blood containing factors A, B and X as described in Example 1.The purified serum may be diluted and used in blood typing or may befirst dried for more convenient handling. If the product is dried, it isusually desired to dilute it with an inert solid diluent which does noteffect the agglutination test as in Example 1.

To use this more potent Rh agglutinin in typing blood it is merelynecessary to prepare a mixture of two drops to the blood to be testedwith onehalf cc. of saline and place one drop of the cell suspension ona glass slide with an equal drop of the typing serum just described. Thetwo drops are mixed and spread out on the slide. Within five minutes astrong clumping action will be easily observed with the naked eye .ifthe blood being tested is Rh positive. Obviously this is a considerableimprovement over the Rh test now' in use.

Blood typing reagents specific to types M and N are prepared exactly asin the preparation of the grouping reagents A and B with the exception,of course, that OM or ON cells are used in immunizing the rabbits, the Xfactor being blocked out in the same way with anti-X.

I claim:

1. The method of producing blood serum containing a high titer of aspecific hemagglutinin, the steps of which comprise obtaining bloodsubstance having hemagglutinogenic properties, mixing therewithhemagglutinins effective against the hemagglutinogenic substances otherthan those of the desired specific hemagglutinin whereby the undesiredhemagglutinogenic substances are rendered substantially impotent tostimulate the production of antibodies, introducing into the bloodstream of a higher animal an effective amount of the thus treatedhemagglutinogenic substance, allowing the formation of correspondinghemagglutins to take place and thereafter removing at least part of theblood of said animal to obtain blood serum containing the desiredhemagglutinin.

2. The method of producing blood serum containing the agglutinin anti-Athe steps which comprise mixing with a blood substance having theagglutinogenic substances A and X a blood substance containing anti-X insufficient amounts to substantially neutralize the agglutinogenic factorX present in said blood substance whereby the agglutinogenic substancesX comprise mixing with a blood substance having containing anti-A.

I 3. The method of producing blood serum containing the agglutininanti-A the steps which the agglutinogenic substances A' and X a bloodsubstance containing anti-X in sufficient amounts to substantiallyneutralize the agglutinogenic factor X present in said blood sub- Ustance whereby the agglutinogenic substances X and thereafter removingat least part of the blood of said animal to obtain blood serumcontaining anti-A.

4. The method of producing blood serum containing the agglutinin anti-Bthe steps which comprise mixing with a blood substance having theagglutinogenic substances B and X a blood substance containing anti-X insufiicient amounts to substantially neutralize the agglutinogenic factorX present in said blood substance whereby the agglutinogenic substancesX are rendered substantially impotent to stimulate the production ofantibodies, introducing into the blood stream of a higher animal aneffective amount of the thus treated agglutinogenic substance, a1-lowing the formation of anti-B to take place and thereafter removing atleast part of the blood of said animal to obtain blood serum containinganti-B.

5. The method of producing blood serum containing the agglutinin anti-Bthe steps which comprise mixing with a blood substance having theagglutinogenic substances B and X a blood substance containing anti-Xinsufficient amounts to substantially neutralize the agglutinogenicfactor X present in said blood substance whereby the agglutinogenicsubstances X are rendered substantially impotent to stimulate theproduction of antibodies, introducing into the blood stream of a rabbitan effective amount of the thus treated agglutinogenic substance, al-

lowing the formation of anti-B to take place and thereafter removing atleast part of the blood of said animal to obtain blood serum containinganti-B.

6. The method of producing blood serum containing the agglutinin anti-Athe steps which comprise introducing into the blood stream of a rabbiterythrocytes of blood of group O and after a suitable concentration ofagglutinin anti-X has been formed therein removing at least part of saidblood, mixing a sufiicient quantity of the anti-X substance thusobtained with erythrocytes of group A to neutralize the antigenicsubstance A contained therein, introducing into the blood stream of arabbit an amount of the thus treated erythrocytes of group A sufficientto produce an immunizing effect, allowing the formation of anti-A totake place in the blood stream of the rabbit and thereafter removing atleast part of the blood of said rabbit to obtain blood serum having arelatively high titer of anti-A.

taining the agglutinin anti-B the steps which comprise introducing intothe blood stream of a rabbit erythrocytes of blood of group O and aftera suitable concentration of agglutinin anti-X has been formed thereinremoving at least part of said blood, mixing a sufficient quantity ofthe anti-X substance thus obtained with erythrocytes of group B toneutralize the antigenic substance X contained therein, introducing intothe blood stream of a, rabbit an amount of the thus treated erythrocytesof group B suflicient to produce an immunizing effect, allowing theformation of anti-B to take place in the blood stream of the rabbit andthereafter removing at least part of the blood of said rabbit to obtainblood serum having a relatively high titer or anti-B.

8. The method of producing blood serum containing the agglutim'n anti-Rhthe steps which comprise introducing into the blood stream of a higheranimal the agglutinogenic substances A, B, and X and, after a suitableconcentration of anti-A, anti-B, and anti-X has been formed in the bloodstream of the animal, removing at least part of said blood, mixing asufficient quantity of the antibodies thus obtained with Rh erythrocytesto neutralize the antigenic substances A, B and X therein, introducinginto the blood stream of a higher animal an amount of the thus treatedRh erythrocytes sufficient to produce an immunizing effect, allowing theformation of anti-Rh to take place in the blood stream of the animal andthereafter removing at least part of the blood of the said animal toobtain a blood serum having a relatively high titer of anti-Rh.

9. The method of producing blood serum containing a high titer of theagglutinin anti-Rh the steps which comprise preparing a blocking serumcontaining the agglutinogens A, B and X, mixing said blocking serum witherythrocytes of the Rhesus monkey to neutralize the antigenic substancesA, B, and X therein, introducing into the blood stream of a goat anamount of the thus treated Rhesus erythrocytes in which the antigenicsubstances A, B and X have been rendered substantially impotent tostimulate the production of antibodies, allowing the formation of anti-Rh to take place in the blood stream of the goat and thereafter removingat least part of the blood of the goat to obtain blood serum having arelatively high titer of anti-Rh.

10. The method which comprises injecting into a warm blooded animalagglutinogens of group X and after agglutinins of group anti-X have beenformed withdrawing at least part of the blood of said animal, mixing thethus produced anti-X with normal erythrocytes to neutralize theagglutinogens of group X therein and injectin the resultingagglutinogenic substance into a warm blooded animal whereby a high titerof agglutinins is produced in the blood serum of the animal.

11. The method which comprises injecting into a warm blooded animalagglutinogens of the group X, A, and B and after agglutinins of thegroup anti-X, anti-A, and anti-B have been formed withdrawing at leastpart of the blood of said animal, mixing the thus produced anti-X, anti-A, and anti-B with Rh erythrocytes to neutralize the agglutinogens X, A,and B therein and injecting the resulting agglutinogenic substance intoa. goat whereby a high titer of Rh agglutinins is produced in the bloodserum of the goat.

ARTHUR F. COCA.

REFERENCES CITED The following references are of record in the file ofthis patent:

Morgan, An Artificial Antigen with Blood- Group A Specificity, in TheBritish Journ. of Experimental Pathology, vol, XXIV, No. 2, April 1943,pages 41-49.

Potent Typing Sera article by Witebsky et al. in Proc. Soc. Exptl. Biol.8: Med, March 1944, pages 167 to 170*.

